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Background: Radiotherapy (RT) can drive cancer cells to enter a state of cellular senescence in which cells can secrete senescence-associated secretory phenotype (SASP) and produce small extracellular vesicles (sEVs) to interact with cells in the tumor microenvironment (TME). Tumor-derived sEVs that are taken up by recipient cells contribute to cancer cell metabolic plasticity, resistance to anticancer therapy, and adaptation to the TME. However, how radiation-induced sEVs support oral squamous cell carcinoma (OSCC) progression remains unclear., Methods: Beta-galactosidase staining and SASP mRNA expression analysis were used to evaluate the senescence-associated activity of OSCC cells after irradiation. Nanoparticle tracking analysis was performed to identify radiation-induced sEVs. Liquid chromatography-tandem mass spectrometry (LC-MS) was used to explore changes in the levels of proteins in radiation-induced sEVs. Cell Counting Kit-8 and colony formation assays were performed to investigate the function of radiation-induced SASP and sEVs in vitro. A xenograft tumor model was established to investigate the functions of radiation-induced sEVs and V-9302 in vivo as well as the underlying mechanisms. Bioinformatics analysis was performed to determine the relationship between glutamine metabolism and OSCC recurrence., Results: We determined that the radiation-induced SASP triggered OSCC cell proliferation. Additionally, radiation-induced sEVs exacerbated OSCC cell malignancy. LC-MS/MS and bioinformatics analyses revealed that SLC1A5, which is a cellular receptor that participates in glutamine uptake, was significantly enriched in radiation-induced sEVs. In vitro and in vivo, inhibiting SLC1A5 could block the oncogenic effects of radiation-induced sEVs in OSCC., Conclusion: Radiation-induced sEVs might promote the proliferation of unirradiated cancer cells by enhancing glutamine metabolism; this might be a novel molecular mechanism underlying radiation resistance in OSCC patients., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Cancer-associated fibroblasts (CAFs) are abundant and heterogeneous in tumor microenvironment (TME). Cross-talk between cancer cells and CAFs results in cancer progression. Here, we demonstrated that a distinct cancer-associated fibroblasts subset with podoplanin (PDPN) positive expression (PDPN + CAFs) was correlated with poor survival in oral squamous cell carcinoma (OSCC). PDPN + CAFs promoted the progression of OSCC by transferring exosomal lncRNA FTX to OSCC cells. Mechanically, FTX bound to flap endonuclease-1 (FEN1), forming an RNA‒protein complex. FTX enhanced promoter demethylation of FEN1 by recruiting ten-eleven translocation-2 (TET2). In addition, FTX/FEN1 axis promoted OSCC cells motility by inhibiting ferroptosis. In xenograft experiments, RSL-3, a ferroptosis-inducing agent, suppressed the tumorigenesis potential of FEN1-overexpressed OSCC cells. Furthermore, Acyl-CoA synthetase long-chain family member 4 (ACSL4) was confirmed to participate in the motility promotion induced by FEN1 overexpression. FEN1 could bind to promoter region of ACSL4 and then inhibit ferroptosis in OSCC cells. Our study reveals that PDPN + CAFs promote the invasiveness of OSCC cells by inhibiting ferroptosis through FTX/FEN1/ACSL4 signaling cascade. PDPN + CAFs may serve as a novel potential therapeutic target for OSCC., (© 2023. The Author(s).)

Background: Long non-coding RNA BRAF-activated non-protein coding RNA plays bidirectional roles in human cancers. However, function and molecular mechanism of BRAF-activated non-protein coding RNA in oral squamous cell carcinoma still need to clarify further., Methods: Long non-coding RNA microarray assay, in situ hybridization staining, clinicopathological data analysis were performed to investigate expression pattern of BRAF-activated non-protein coding RNA in oral squamous cell carcinoma tissue samples. Constructing ectopically expressed BRAF-activated non-protein coding RNA in oral squamous cell carcinoma cells via plasmids or siRNAs, then changeable abilities of proliferation and motility of these cells were observed in vitro and in vivo. RNA-protein pulldown, RNA immunoprecipitation, and bioinformatics analyses were performed to explore potential pathways involved in BRAF-activated non-protein coding RNA-based regulation of malignant progression in oral squamous cell carcinoma., Results: BRAF-activated non-protein coding RNA was identified upregulated in oral squamous cell carcinoma tissue and correlated with nodal metastasis and clinical severity of patients. Overexpressed BRAF-activated non-protein coding RNA increased percentage of 5-ethynyl-2'-deoxyuridine-positive cells, viability, migration, and invasion rates of oral squamous cell carcinoma cells, while silenced BRAF-activated non-protein coding RNA could observe weakened effects in vitro. Xenograft tumor formed by BRAF-activated non-protein coding RNA-overexpressed cells had bigger volume, faster growth rates, higher weight, and more Ki67 + cells. Pulmonary metastasis induced by BRAF-activated non-protein coding RNA-silenced cells had fewer colony nodes, Ki67 + cells, and CD31 + blood vessels. Furthermore, BRAF-activated non-protein coding RNA was mainly localized in nucleus of oral squamous cell carcinoma cells and bound Ras-associated binding 1A. Silencing Ras-associated binding 1A could damage mobile ability and phosphorylation levels of nuclear factor-κB in oral squamous cell carcinoma cells induced by overexpressing BRAF-activated non-protein coding RNA. Opposite trend was also observed., Conclusion: Acting as a promoter in oral squamous cell carcinoma metastasis, BRAF-activated non-protein coding RNA promotes oral squamous cell carcinoma cells proliferation and motility by regulating the BRAF-activated non-protein coding RNA/Ras-associated binding 1A complex, which activates nuclear factor-κB signaling pathway., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objectives: Cancer-associated fibroblasts (CAFs) are vital constituents of the tumor microenvironment (TME) and play a predominant role in oral squamous cell carcinoma (OSCC) progression. We aimed to investigate the effect and mechanism of exosomal miR-146b-5p derived from CAFs on the malignant biological behavior of OSCC., Materials and Methods: Illumina small RNA (sRNA) sequencing was conducted to determine the differential expression patterns of microRNAs (miRNAs) in exosomes derived from CAFs and normal fibroblasts (NFs). Transwell and cell counting kit-8 (CCK-8) assays and xenograft tumor models in nude mice were used to investigate the effect of CAF exosomes and miR-146b-p on the malignant biological behavior of OSCC. Reverse transcription quantitative real-time PCR (qRT-PCR), luciferase reporter, western blotting (WB) and immunohistochemistry assays were employed to investigate the underlying mechanisms involved in CAF exosomes that promote OSCC progression., Results: We demonstrated that CAF-derived exosomes were taken up by OSCC cells and enhanced the proliferation, migration, and invasion ability of OSCC. Compared with NFs, the expression of miR-146b-5p was increased in exosomes and their parent CAFs. Further studies showed that the decreased expression of miR-146b-5p inhibited the proliferation, migration and invasion ability of OSCC cells in vitro and the growth of OSCC cells in vivo. Mechanistically, miR-146b-5p overexpression led to the suppression of HIKP3 by directly targeting the 3'-UTR of HIPK3, as confirmed by luciferase assay. Reciprocally, HIPK3 knockdown partially reversed the inhibitory effect of the miR-146b-5p inhibitor on the proliferation, migration, and invasion ability of OSCC cells and restored their malignant phenotype., Conclusions: Our results revealed that CAF-derived exosomes contained higher levels of miR-146b-5p than NFs, and miR-146b-5p overexpression in exosomes promoted the malignant phenotype of OSCC by targeting HIPK3. Therefore, inhibiting exosomal miR-146b-5p secretion may be a promising therapeutic modality for OSCC., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Oral squamous cell carcinoma (OSCC), with aggressive locoregional invasion, has a high rate of early recurrences and poor prognosis. Dihydroartemisinin (DHA), as a derivative of artemisinin, has been found to exert potent antitumor activity. Recent studies reported that DHA suppresses OSCC cell growth and viability through the regulation of reactive oxygen species (ROS) production and mitochondrial calcium uniporter. However, the mechanism underlying the action of DHA on OSCCs remains elusive. In the study, we observed that 159 genes were remarkably misregulated in primary OSCC tumors associated with DHA-inhibited pathways, supporting that OSCCs are susceptible to DHA treatment. Herein, our study showed that DHA exhibited promising effects to suppress OSCC cell growth and survival, and single-cell colony formation. Interestingly, the combination of DHA and cisplatin (CDDP) significantly reduced the toxicity of CDDP treatment alone on human normal oral cells (NOK). Moreover, DHA remarkably impaired mitochondrial structure and function, and triggered DNA damage and ROS generation, and activation of mitophagy. In addition, DHA induced leakage of cytochrome C and apoptosis-inducing factor (AIF) from mitochondria, elevated Bax/cleaved-caspase 3 expression levels and compromised Bcl2 protein expression. In the OSCC tumor-xenograft mice model, DHA remarkably suppressed tumor growth and induced apoptosis of OSCCs in vivo . Intriguingly, a selective mitophagy inhibitor Mdivi-1 could significantly reinforce the anticancer activity of DHA treatment. DHA and Mdivi-1 can synergistically suppress OSCC cell proliferation and survival. These data uncover a previously unappreciated contribution of the mitochondria-associated pathway to the antitumor activity of DHA on OSCCs. Our study shed light on a new aspect of a DHA-based therapeutic strategy to combat OSCC tumors., Competing Interests: All authors declare that they have no conflict of interest., (Copyright © 2023 Shanwei Shi et al.)

Oral squamous cell carcinoma (OSCC) is the 11th most prevalent tumor worldwide. Despite advantages of therapeutic approaches, the 5-year survival rate of patients with OSCC is less than 50%. It is urgent to elucidate mechanisms underlying OSCC progression for developing novel treatment strategies. Our recent study has revealed that Keratin 4 (KRT4) suppresses OSCC development, which is downregulated in OSCC. Nevertheless, the mechanism downregulating KRT4 in OSCC remains unknown. In this study, touchdown PCR was utilized to detect KRT4 pre-mRNA splicing, while m 6 A RNA methylation was identified by methylated RNA immunoprecipitation (MeRIP). Besides, RNA immunoprecipitation (RIP) was used to determine RNA-protein interaction. Herein, this study indicated that intron splicing of KRT4 pre-mRNA was suppressed in OSCC. Mechanistically, m 6 A methylation of exon-intron boundaries prevented intron splicing of KRT4 pre-mRNA in OSCC. Besides, m 6 A methylation suppressed the binding of splice factor DGCR8 microprocessor complex subunit (DGCR8) to exon-intron boundaries in KRT4 pre-mRNA to prohibit intron splicing of KRT4 pre-mRNA in OSCC. These findings revealed the mechanism downregulating KRT4 in OSCC and provided potential therapeutic targets for OSCC., Competing Interests: The authors declare there are no competing interests., (©2023 Li et al.)

Objective: Oral squamous cell carcinoma (OSCC) is characterized by high recurrence and metastasis and places a heavy burden on societies worldwide. Cancer cells thrive in a changing microenvironment by reprogramming lipidomic metabolic processes to provide nutrients and energy, activate oncogenic signaling pathways, and manage redox homeostasis to avoid lipotoxicity. The mechanism by which OSCC cells maintain lipid homeostasis during malignant progression is unclear., Methods: The altered expression of fatty acid (FA) metabolism genes in OSCC, compared with that in normal tissues, and in OSCC patients with or without recurrence or metastasis were determined using public data from the TCGA and GEO databases. Immunohistochemistry was performed to examine the carboxylesterase 2 (CES2) protein level in our own cohort. CCK-8 and Transwell assays and an in vivo xenograft model were used to evaluate the biological functions of CES2. Mass spectrometry and RNA sequencing were performed to determine the lipidome and transcriptome alterations induced by CES2. Mitochondrial mass, mtDNA content, mitochondrial membrane potential, ROS levels, and oxygen consumption and apoptosis rates were evaluated to determine the effects of CES2 on mitochondrial function in OSCC., Results: CES2 was downregulated in OSCC patients, especially those with recurrence or metastasis. CES2 high OSCC patients showed better overall survival than CES2 low OSCC patients. Restoring CES2 expression reduced OSCC cell viability and suppressed their migration and invasion in vitro, and it inhibited OSCC tumor growth in vivo. CES2 reprogrammed lipid metabolism in OSCC cells by hydrolyzing neutral lipid diacylglycerols (DGs) to release free fatty acids and reduce the membrane structure lipid phospholipids (PLs) synthesis. Free FAs were converted to acyl-carnitines (CARs) and transferred to mitochondria for oxidation, which induced reactive oxygen species (ROS) accumulation, mitochondrial damage, and apoptosis activation. Furthermore, the reduction in signaling lipids, e.g., DGs, PLs and substrates, suppressed PI3K/AKT/MYC signaling pathways. Restoring MYC rescued the diminished cell viability, suppressed migratory and invasive abilities, damaged mitochondria and reduced apoptosis rate induced by CES2., Conclusions: We demonstrated that CES2 downregulation plays an important role in OSCC by maintaining lipid homeostasis and reducing lipotoxicity during tumor progression and may provide a potential therapeutic target for OSCC., Competing Interests: Conflict of Interest None declared., (Copyright © 2022 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Although obesity has been associated with an increased risk and aggressiveness of many types of carcinoma, whether it promotes squamous cell carcinoma remains unclear. To reveal the role of obesity in oral squamous cell carcinoma (OSCC) initiation and development, we used 4NQO-induced OSCC model mice to examine the impact of dietary obesity on carcinogenesis. The results showed that high-fat diet (HFD)-induced obesity significantly promoted the incidence of OSCC and altered the local immune microenvironment with the expansion of CD11b + Gr1 + myeloid-derived suppressor cells (MDSCs). The underlying mechanism that induced an immunosuppressive local microenvironment in obesity was the recruitment of MDSCs through the CCL9/CCR1 axis and enhancement of MDSC immunosuppressive function via intracellular fatty acid uptake. Furthermore, clinical samples verified the increase in infiltrated CD33 + (a marker of human MDSCs) cells in obese OSCC patients, and data from the TCGA dataset confirmed that CD33 expression was positively correlated with local adipocytes in OSCC. Survival analysis showed that enrichment of adipocytes and high expression of CD33 were associated with poor prognosis in OSCC patients. Strikingly, depletion of MDSCs significantly ameliorated HFD-promoted carcinogenesis in 4NQO-induced model mice. These findings indicate that obesity is also an important risk factor for OSCC, and cancer immunotherapy, especially targeting MDSCs, may exhibit greater antitumor efficacy in obese patients., (© 2021. The Author(s).)

Tacrolimus (TAC, FK506) is a major calcineurin inhibitor and has been commonly used in treatments of patients with organ transplants and immune diseases. Moreover, tacrolimus is recommended by the treatment guidelines for oral potentially malignant disorders (OPMDs) such as oral lichen planus (OLP). However, whether tacrolimus increases the risk of cancer remains controversial. We observed that in a 4-Nitroquinoline N-oxide (4NQO)-induced oral carcinogenesis model, tacrolimus treatment was associated with a significantly lower ratio of cancer formation (52.94% vs. 90%) and a lower proportion of Ki67 and proliferation cell nuclear antigen (PCNA) -positive cells in lesion areas (P < 0.001). Liver, kidney, and lung functions of rats and the tumor immune microenvironment of the tongue were not affected. These observations suggest that tacrolimus blocked oral carcinogenesis through epithelial cell proliferation inhibition, independent of its immunosuppressive effects. As a processing factor, tacrolimus decreased tumor formation and cell proliferation in different stages of oral squamous cell carcinoma (OSCC) progression in vivo and in vitro. Furthermore, we investigated effects on the cell cycle and expression of related proteins. Tacrolimus induced G1/S phase arrest and significantly downregulated the expression of cyclinD1, cyclinE1, and c-Myc. These results suggest that tacrolimus induces G1/S phase arrest via inhibition of cyclinD1, cyclinE1, and c-Myc expression and retards oral cell carcinogenesis in vitro and in vivo. Thus, application of tacrolimus is a safe therapeutic strategy for treating OPMDs., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Oral squamous cell carcinoma (OSCC) is the major cause of morbidity and mortality in head and neck cancer patients worldwide. This malignant disease is challenging to treat because of the lack of effective curative strategies and the high incidence of recurrence. This study aimed to investigate the efficacy of a single and dual approach targeting ribosome biogenesis and protein translation to treat OSCC associated with the copy number variation (CNV) of ribosomal DNA (rDNA). Here, we found that primary OSCC tumors frequently exhibited a partial loss of 45S rDNA copy number and demonstrated a high susceptibility to CX5461 (a selective inhibitor of RNA polymerase I) and the coadministration of CX5461 and INK128 (a potent inhibitor of mTORC1/2). Combined treatment displayed the promising synergistic effects that induced cell apoptosis and reactive oxygen species (ROS) generation, and inhibited cell growth and proliferation. Moreover, INK128 compromised NHEJ-DNA repair pathway to reinforce the antitumor activity of CX5461. In vivo, the cotreatment synergistically suppressed tumor growth, triggered apoptosis and strikingly extended the survival time of tumor-bearing mice. Additionally, treatment with the individual compounds and coadministration appeared to reduce the incidence of enlarged inguinal lymph nodes. Our study supports that the combination of CX5461 and INK128 is a novel and efficacious therapeutic strategy that can combat this cancer and that 45S rDNA may serve as a useful indicator to predict the efficacy of this cotreatment., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Objectives: miRNAs in salivary exosomes are used as novel non-invasive biomarkers for detection strategies of human disease. Here, we aimed to investigate the diagnostic potential of salivary exosomal miRNAs as biomarkers for screening oral squamous cell carcinoma (OSCC) and to explore the underlying mechanisms of OSCC pathogenesis., Materials and Methods: Differentially expressed miRNAs were obtained from salivary exosomes of four healthy controls and four OSCC patients using miRNA microarray analysis. The expression of miR-24-3p in the salivary exosomes was then verified by qRT-PCR. The diagnostic power was assessed by receiver operating characteristic (ROC) analysis. Cell proliferation was measured using CCK-8 cell viability assay and colony formation assay. The target gene of miR-24-3p was confirmed by dual luciferase reporter assay., Results: A total of 109 miRNAs were found to be more than 2-fold altered in the salivary of patients and healthy individuals by miRNA microarray. qRT-PCR analysis further confirmed a significant increase of miR-24-3p in the salivary exosomes from 45 preoperative OSCC patients compared to 10 normal controls. ROC analysis showed that miR-24-3p has excellent diagnostic accuracy for OSCC (area under the ROC curve [AUC] = 0.738; P =  0.02). Similarly, we found that miR-24-3p expressed a higher level in OSCC neoplastic tissues, suggesting that circulating miR-24-3p may originate from tumor cells. Furthermore, exogenous exosomal miR-24-3p increased proliferation of recipient malignant cells. Additionally, overexpression of miR-24-3p promoted the proliferation of OSCC cells and regulated the expression of cell cycle-related genes. Dual luciferase reporter assay indicated that miR-24-3p can interact with PER1 directly., Conclusions: Salivary exosomal miR-24-3p is a potential novel diagnostic biomarker for OSCC, and miR-24-3p can maintain the proliferation of OSCC cells through targeting PER1., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Treatment of oral squamous cell carcinoma (OSCC) remains a challenge because of the lack of effective early treatment strategies and high incidence of relapse. Here, we showed that combined 4SC-202 (a novel selective class I HDAC inhibitor) and INK128 (a selective mTORC1/C2 inhibitor) treatment exhibited synergistic effects on inhibiting cell growth, sphere-forming ability, subcutaneous tumor formation and ALDH1 + cancer stem cells (CSCs) in OSCC. The initiation of OSCC was significantly inhibited by combined treatment in 4NQO-induced rat model. In addition, upregulated SOX2 was associated with advanced and metastatic tumors in OSCC patients and was responsible for the drug-resistance property of OSCC cells. The inhibitory effect of combined treatment on cell viability and ALDH1 + CSCs were attenuated by SOX2 verexpression. Furthermore, combined treatment can effectively overcome chemoresistance and inhibit the growth of recurrent OSCC in vitro and in vivo. Mechanistically, 4SC-202 and INK128 repressed SOX2 expression through miR-429/miR-1181-mediated mRNA degradation and preventing cap-dependent mRNA translation, respectively. These results suggest that combined class I histone deacetylase and mTORC1/C2 inhibition suppresses the carcinogenesis and recurrence of OSCC by repressing SOX2., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Oral squamous cell carcinoma (OSCC) is the most common and aggressive epithelial tumor in the head and neck region with a rising incidence. Despite the advances in basic science and clinical research, the overall survival rate of OSCC remains low. Thus finding novel effective therapeutic agents for OSCC is necessary. In this study, we investigated the effects and mechanisms of combined metformin and 4SC-202 in OSCC. Our results showed that metformin and 4SC-202 synergistically suppressed the proliferation and promoted the intrinsic apoptosis of OSCC cells in vitro and in vivo. Importantly, the proteasome inhibitor MG132 impeded the ΔNp63-decreasing effects after metformin and 4SC-202 treatment, indicating that metformin and 4SC-202 could promote the degradation of ΔNp63 protein. Moreover, ubiquitination level of ΔNp63 increased after metformin or/and 4SC-202 administration. Furthermore, we revealed that ΔNp63 mediated anticancer effects of metformin and 4SC-202, as overexpression or suppression of ΔNp63 could attenuate or facilitate the apoptosis rate of OSCC under metformin or/and 4SC-202 treatment. Collectively, metformin and 4SC-202 synergistically promote intrinsic apoptosis through accelerating ubiquitin-mediated degradation of ΔNp63 in OSCC, and this co-treatment can serve as a potential therapeutic scheme for OSCC., (© 2019 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background: Although a few studies suggested that the chemokine CCL2 might be involved in the development of oral squamous cell carcinoma (OSCC), the exact mechanism remains unclear. In this study, we aimed to determine the resource of CCL2 in lesions and explored a potential mechanism that CCL2 promotes tumor progression. The study was an effort to provide new insights into the pathological role of CCL2 in OSCC., Methods: Specimens of OSCC and normal oral mucosa were stained using immunohistochemistry (IHC) to assess the CCL2 expression. Enzyme-linked immunosorbent assay (ELISA) was used to detect the difference of CCL2 between OSCC and normal oral mucosa cell lines. In addition, we treated OSCC cells with exogenous rCCL2 combined with or without CCL2 neutralizing antibody and then determined the changes of in epithelial-mesenchymal transition (EMT) markers and cell migration capacity using immunofluorescence, Western blotting, transwell migration, and wound healing assays., Results: We have found that CCL2 expression was upregulated significantly in both lesions and cell culture supernatant of OSCC compared with controls. IHC staining demonstrated that CCL2 expression was primarily located in the cytoplasm and cell membrane of cells. We have also found that rCCL2 could effectively induce EMT through upregulating Snail in OSCC cells, which was demonstrated by the decrease of E-cadherin and the increase of vimentin. In addition, we have found that CCL2 neutralizing antibody could block EMT induced by CCL2 in OSCC., Conclusions: CCL2 secreted by cancer cells can promote cell migration by inducing EMT via paracrine or autocrine in OSCC., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Recent evidences suggested that Mesenchymal stem cells (MSCs) may be involved in tumor formation by modulating of the tumor microenvironment, but it is still unclear the potential of MSCs in the malignant transformation of oral mucosa. Using a chemically-induced oral carcinogenesis model by 4-nitroquinoline-1-oxide (4NQO), we generated precancerous lesions and cancerous lesions in the oral cavity of rats. Flow cytometric analysis on lesions derived single cell suspension revealed an increase in the proportion of MSCs and a decreased proportion of T cell during oral mucosa malignancy. Moreover, MSCs showed increased immunosuppression capacity on T cell proliferation during mucosa malignancy. At last, we demonstrated that higher frequency of lesions resident MSCs was correlated with more Ki67 expression in the lesion, which indicated higher cellular proliferative status in the lesions. Our study demonstrated that MSCs may play an important role in oral mucosa malignant transformation through regulating T cell proliferation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Elucidation of the mechanisms of T-cell-mediated antitumor responses will provide information for the rational design and development of cancer immunotherapies. Here, we found that calnexin, an endoplasmic reticulum (ER) chaperone protein, is significantly upregulated in oral squamous cell carcinoma (OSCC). Upregulation of its membranous expression on OSCC cells is associated with inhibited T-cell infiltration in tumor tissues and correlates with poor survival of patients with OSCC. We found that calnexin inhibits the proliferation of CD4 + and CD8 + T cells isolated from the whole blood of healthy donors and patients with OSCC and inhibits the secretion of IFNγ, TNFα, and IL2 from these cells. Furthermore, in a melanoma model, knockdown of calnexin enhanced the infiltration and effector functions of T cells in the tumor microenvironment and conferred better control of tumor growth, whereas treatment with a recombinant calnexin protein impaired the infiltration and effector functions of T cells and promoted tumor growth. We also found that calnexin enhanced the expression of PD-1 on CD4 + and CD8 + T cells by restraining the DNA methylation status of a CpG island in the PD-1 promoter. Thus, this work uncovers a mechanism by which T-cell antitumor responses are regulated by calnexin in tumor cells and suggests that calnexin might serve as a potential target for the improvement of antitumor immunotherapy., (©2018 American Association for Cancer Research.)

The dysregulation of glycolysis has been suggested to lead to alteration of cell drug resistance signals, proliferation and metastasis. Emerging evidence indicates that lncRNAs play a key role in the cellular processes of tumor cells, including glycolysis, growth, and movement. However, the role and potential mechanism of lncRNAs in glycolysis-mediated metastasis has not been explored. In this study, we identified a novel lncRNA lnc-p23154 which is associated with OSCC patient metastasis and the promotion of OSCC cell migration and invasion in vitro and in vivo. Furthermore, we found that lnc-p23154 also participates in OSCC glycolysis by facilitating Glut1 expression. Rescue of lnc-p23154 reversed the suppression of OSCC cell migration and invasion induced by Glut1 knockdown. In addition, lnc-p23154 is mainly located in the nucleus and binds to the promoter region of miR-378a-3p, which represses Glut1 expression by targeting to its 3'UTR directly. Therefore, we concluded that lnc-p23154 may play an important role in Glut1-mediated glycolysis by inhibiting miR-378a-3p transcription and accelerate OSCC metastasis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Thrombomodulin (TM, also known as CD141), which functions as an anticoagulant, is widely expressed on cell surface of a variety of cell types, including human blood cells as well as certain immune cells. To determine whether TM could be a potential marker for OSCC diagnosis as well as a molecular target for OSCC therapy, we examined the expression of TM in an oral cancer tissue microarray with 153 oral cancer tissues. Further, we also analyzed the expression of TM on DCs of 36 OSCC patients and 36 healthy donors. The expression of TM was determined using standard immunohistochemistry on a tissue microarray of 153 OSCC patients. Flow cytometric analyses were performed to determine the proportions of CD141 + DCs in the PBMC of 36 OSCC patients and 36 healthy donors. Clinicopathological correlations were performed based on the available clinical data. Our results showed that in the univariate analysis, high TM expression was significantly associated with well differentiation of tumor cells (P=.001), but not correlated with overall survival and disease-free survival (P>.05). In addition, CD141 + DCs were both present in OSCC patients and healthy donors with about 0.04%. There was no significant difference with the percentages of CD141 + DCs in the PBMC of OSCC patients and that of the normal control group (P>.05). This study indicates that TM expression might play the most critical role in the differentiation of OSCC tumors. Functional distinctions of CD141 + DCs in OSCC patients deserve further investigation to provide important therapeutic understandings for future immunotherapy., (Copyright © 2018. Published by Elsevier Inc.)

Oridonin, a bioactive diterpenoid purified from Rabdosia rubescens, has been shown to possess anticancer capacity in several cancer types. However, its effects on oral squamous cell carcinoma (OSCC) cells remain unclear. This study aimed to investigate the anticancer ability of oridonin in OSCC cells, including proliferation, apoptosis and underlying mechanisms using the OSCC cell lines, UM1 and SCC25. The results showed that oridonin not only inhibited proliferation and clonal formation but also induced G2/M cell cycle arrest and apoptosis in UM1 and SCC25 cells in a dose-dependent manner. Western blot revealed that oridonin treatment increased the ratio of Bax/Bcl-2, and activated the cleavage of caspase-3, caspase-9 and PARP-1. Oridonin also induced G2/M phase arrest in OSCC cells via down-regulating the G2/M transition-related proteins such as cyclin B1 or up-regulating cyclin D1, cyclin D3, P21, p-CDK1 and cyclin A2. In addition, oridonin treatment significantly inhibited the phosphorylation of PI3K and Akt and inhibited tumor growth of OSCC xenograft in nude mice. Taken together, these results suggested that oridonin possesses anti-oral cancer capacity via inhibiting the PI3K/Akt signaling and induce apoptosis and G2/M-phase arrest. Therefore, oridonin may be a potential anticancer drug for the treatment of oral cancer., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

The majority of cases of oral squamous cell carcinoma (OSCC) develop from oral potentially malignant disorders, which have been confirmed to be involved in chronic oxidative stimulation. However, no effective treatment approaches have been used to prevent the development of dysplasia into cancerous lesions thus far. In the present study, a well-established OSCC model was used to detect proteomics profiles at different stages during oral malignant transformation. Of the 15 proteins that were found to be upregulated in both the dysplasia and carcinoma stages, the oxidative stress-associated proteins, thioredoxin-1 (Trx-1), glutaredoxin-1 and peroxiredoxin-2 were note as the proteins with significant changes in expression Trx-1 was identified to be the most significantly upregulated protein in the precancerous stage. Validation experiments confirmed that Trx-1 was overexpressed both in dysplasia and cancerous tissue samples, and the inhibition of Trx-1 was able to promote the apoptosis of OSCC cells under hypoxic conditions. Furthermore, the experimental application of a Trx-1-specific inhibitory agent in an animal model led to a lower cancerization rate and a delay in tumor formation. The possible mechanisms were associated with the increased apoptosis via a reactive oxygen species (ROS)-dependent pathway. Taken together, our findings indicate that Trx-1 may be an important target for delaying oral malignant transformation, which provides a novel therapeutic strategy for the prevention and treatment of OSCC.

Stromal-epithelial lactate shuttle is an essential process to support fast‑growing tumor cells, however, the underlying mechanism remains ambiguous. Interleukin‑1β (IL‑1β), which is a key node gene in both stromal and epithelial cells of oral squamous cell carcinoma (OSCC), may participate in this metabolic reprogramming. In the present study, anaerobic glycolysis of cancer‑associated fibroblasts (CAFs) was evaluated and the role of IL‑1β in regulating stromal‑epithelial lactate shuttle was determined. A co‑culture system of primary fibroblasts and OSCC cell lines (CAL27, UM1 or SCC25) was created to investigate the stromal‑epithelial interaction. α‑smooth muscle actin (α‑SMA) expression of fibroblasts, IL‑1β expression and cell proliferation of OSCC cells, and a series of glycolytic genes were measured. Recombinant IL‑1β treatment and IL‑1β knockdown in UM1 cells were also used to evaluate the effect of IL‑1β. Expression of α‑SMA, glucose transporter 1, hexokinase 2, lactic dehydrogenase and mono‑carboxylate transporter (MCT) 4 were significantly overexpressed in activated fibroblasts, while IL‑1β and MCT1 were upregulated in OSCC cells, indicating enhanced glycolysis in cells of the tumor stroma and a lactate shuttle to the tumor cells. Furthermore, exogenous IL‑1β induced fibroblasts to present similar expression profiles as that in the co‑culture system. Silencing of IL‑1β significantly abrogated the regulatory effect of UM1 cells on stromal glycolysis. Additionally, carboxy‑fluorescein succinimidyl ester cell tracing indicated that OSCC cell proliferation was accelerated during co‑cultivation with fibroblasts. These results indicate that tumor‑derived IL‑1β enhanced stromal glycolysis and induced one‑way lactate flow from the tumor mesenchyme to transformed epithelium, which promotes OSCC proliferation.

Chemokine (CC motif) ligand 18 (CCL18) is involved in remodeling of the tumor microenvironment and plays critical roles in oncogenesis, invasiveness, and metastasis. We previously investigated the overexpression of CCL18 in primary oral squamous cell carcinoma (OSCC) tissues and its association with advanced clinical stage in OSCC patients. However, the underlying mechanisms of this CCL18-derived activity remains unidentified. This study showed exogenous CCL18 increased cell migration and invasion and induced cell epithelial-mesenchymal transition (EMT), and that E-cadherin, an epithelial marker, decreased and N-cadherin, a mesenchymal marker, increased, compared to negative control in OSCC cells. Furthermore, we detected that CCL18 induced the acquisition of cancer stem(-like) cell characteristics in oral cancer cells, but also found a significantly positive correlation between the expression of CCL18 and Bmi-1 (P < 0.001) in OSCC surgical specimens by immunohistochemistry analysis. The expression of octamer-binding transcription factor 4 and Bmi-1 were significantly upregulated, and proportions of aldehyde dehydrogenase high+ cells and CD133 + cells were markedly increased in CCL18-treated cells compared to untreated cells. Sphere formation ability was observably enhanced when cells were continually exposed to high levels of CCL18. Moreover, CCL18 upregulated Slug expression by stimulating the mammalian target of rapamycin (mTOR) signaling pathway in OSCC cell lines. Inhibition of the mTOR pathway by INK128, or Slug knockdown by RNA interference, reversed CCL18-induced EMT and the stemness response at both molecular and functional levels. In conclusion, our data suggested that CCL18 upregulated Slug expression to promote EMT and stem cell-like features by activating the mTOR pathway in oral cancer. These findings provide new potential targets for the early diagnosis and treatment of OSCC., (© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background: Prognostic factors aid in the stratification and treatment of cancer. This study evaluated prognostic importance of tumor infiltrating immune cell in patients with oral squamous cell carcinoma., Methods: Profiles of infiltrating immune cells and clinicopathological data were available for 78 OSCC patients with a median follow-up of 48 months. The infiltrating intensity of CD8, CD4, T-bet, CD68 and CD57 positive cells were assessed by immunohistochemistry. Chi-square test was used to compare immune markers expression and clinicopathological parameters. Univariate and multivariate COX proportional hazard models were used to assess the prognostic discriminator power of immune cells. The predictive potential of immune cells for survival of OSCC patients was determined using ROC and AUC., Results: The mean value of CD8, CD4, T-bet, CD68 and CD57 expression were 28.99, 62.06, 8.97, 21.25 and 15.75 cells per high-power field respectively. The patient cohort was separated into low and high expression groups by the mean value. Higher CD8 expression was associated with no regional lymph node metastasis (p = 0.033). Patients with more abundant stroma CD57 + cells showed no metastasis into regional lymph node (p = 0.005), and early clinical stage (p = 0.016). The univariate COX regression analyses showed that no lymph node involvement (p < 0.001), early clinical stage (TNM staging I/II vs III/IV, p = 0.007), higher CD8 and CD57 expression (p < 0.001) were all positively correlated with longer overall survival. Multivariate COX regression analysis showed that no lymph node involvement (p = 0.008), higher CD8 (p = 0.03) and CD57 (p < 0.001) expression could be independent prognostic indicators of better survival. None of CD4, T-bet or CD68 was associated with survival in ether univariate or multivariate analysis. ROC and AUC showed that the predictive accuracy of CD8 and CD57 were all superior compared with TNM staging. CD57 (AUC = 0.868; 95% CI, 0.785-0.950) and CD8 (AUC = 0.784; 95% CI, 0.680-0.889) both provided high predictive accuracy, of which, CD57 was the best predictor., Conclusion: Tumor stroma CD57 and CD8 expression was associated with lymphnode status and independently predicts survival of OSCC patients. Our results suggest an active immune microenvironment in OSCC that may be targetable by immune drugs.

Chemokine (C-C motif) ligand 18 (CCL18) has been implicated in the pathogenesis and progression of various cancers; however, in oral squamous cell carcinoma (OSCC), the role of CCL18 is unknown. In this study, we found that CCL18 was overexpressed in primary OSCC tissues and was associated with an advanced clinical stage. CCL18 was found in both the cytoplasm and cell membrane of OSCC cells and was predominantly produced by cancer epithelial cells, as opposed to tumor-infiltrating macrophages. In vitro studies indicated that the effects of endogenous CCL18 on OSCC cell growth, migration, and invasion could be blocked by treatment with a neutralizing anti-CCL18 antibody or CCL18 knockdown, while exogenous recombinant CCL18 (rCCL18) rescued those effects. Akt was activated in rCCL18-treated OSCC cells, while LY294002, a pan-PI3K inhibitor, abolished both endogenous and exogenous CCL18-induced OSCC cell invasion. In vivo, LY294002 treatment attenuated rCCL18-induced OSCC cell growth. Our results indicate that CCL18 acts in an autocrine manner via Akt activation to stimulate OSCC cell growth and invasion during OSCC progression. They also provide a potential therapeutic target for the treatment of oral cancer.

The chemokine CXCL12 and its receptors CXCR4 and CXCR7 might play important roles in the occurrence and development of oral squamous cell carcinoma (OSCC). While CXCR4 expression is associated to initiation and progression of OSCC, the role of CXCR7, the recently founded second CXCL12 receptor, has not yet been elucidated in OSCC. In this study, CXCR4 and CXCR7 expressions were evaluated using western blot and quantitative RT-PCR in OSCC cells. AMD3100 (CXCR4 antagonist) was used to inhibit the activation of CXCR4. In contrast to CXCR4, effective CXCR7 small interfering RNA (siRNA) segments were used to silence CXCR7 in OSCC cells. The biological effects of CXCL12-CXCR4/CXCR7 axis on OSCC cell lines were studied by CCK-8 and transwell assay. As determined by RT-PCR and Western blot, CXCR7 expression was significantly downregulated after siRNA transfection in OSCC cells, and thus significantly promoted OSCC cell migration and invasion in vitro. The relative roles of the two CXCL12 receptors were further assessed by CXCR7 knockdown or deactivate CXCR4 receptor alone, or in combination, in the OSCC cells. In vitro functional analyses indicated that, in response to their common ligand (CXCL12), both receptors induced inhibition of proliferation and migration in OSCC cells in a dose-dependent manner. Exogenous CXCL12 could promote cell migration and invasion. In conclusion, our results indicated that CXCL12 which combined its receptor CXCR4 and CXCR7 together could promote cell motilities of OSCC.

Background: Clotrimazole is an antifungal imidazole derivative showing anti- neoplastic effect in some tumors, but its anticancer potential is still unclear in oral squamous cell carcinoma (OSCC). The aim of this study was to evaluate the antitumor effect of clotrimazole, and to investigate the possible mechanism of clotrimazole-mediated antitumor activity in OSCC., Methodology: In vitro experiments, the cell viability and clonogenic ability of three human OSCC cell lines CAL27, SCC25 and UM1 were detected after clotrimazole treatment by CCK8 assay and colony formation assay. Cell cycle progression and apoptosis were assessed by flow cytometry, and the involvement of several mediators of apoptosis was examined by western blot analysis. Then, the in vivo antitumor effect of clotrimazole was investigated in CAL27 xenograft model. Immunohistochemistry and western blot analysis were performed to determine the presence of apoptotic cells and the expression of Bcl-2 and Bax in tumors from mice treated with or without clotrimazole., Results: Clotrimazole inhibited proliferation in all three OSCC cell lines in a dose-and time-dependent manner, and significantly reduced the colony formation of OSCC cells in vitro. Clotrimazole caused cell cycle arrest at the G0/G1 phase. In addition, clotrimazole induced apoptosis in OSCC cells, and significantly down-regulated the anti-apoptotic protein Bcl-2 and up-regulated the pro-apoptotic protein Bax. Notably, clotrimazole treatment inhibited OSCC tumor growth and cell proliferation in CAL27 xenograft model. Clotrimazole also markedly reduced Bcl-2 expression and increased the protein level of Bax in tumor tissues of xenograft model., Conclusion: Our findings demonstrated a potent anticancer effect of clotrimazole by inducing cell cycle arrest and cellular apoptosis in OSCC.

Objective: All-trans retinoic acid (ATRA) has been demonstrated to inhibit tumor growth by restoration of gap junctional intercellular communication (GJIC) via upregulation of connexin (Cx) expression in some solid tumors. However, the relationship between ATRA and GJIC remains unclear in oral squamous cell carcinoma (OSCC). The aim of this study was to investigate the effect of ATRA on the GJIC function of OSCC., Study Design: We measured the effects of ATRA on the viability and cell cycle distribution of SCC9 and Tca8113 OSCC cells. The GJIC function was observed using the scrape-loading dye transfer technique, and the mRNA and protein levels of Cx32 and Cx43 were detected by qRT-PCR, Western blot, and immunofluorescence assays., Results: ATRA inhibited the growth of OSCC cells in a dose- and time-dependent manner (P <0.05) and caused cell cycle arrest. ATRA-treated cells showed a 2.69-fold and 2.06-fold enhancement of GJIC in SCC9 and Tca8113 cells, respectively (P <0.05). Moreover, ATRA induced upregulation of Cx32 and Cx43 at both the mRNA and protein levels in OSCC cells., Conclusion: Our results indicated that restoration of GJIC via enhanced Cx32 and Cx43 expression might serve as a novel mechanism for the anti-tumor effect of ATRA in OSCC.

Objective: The chemokine receptor CXCR4 and its ligand CXCL12 have been suggested to play important roles in the initiation or progression of cancers. The goal of the present study was to investigate alterations of CXCL12/CXCR4 in oral premalignant lesions and oral squamous cell carcinoma (OSCC)., Methods: In 13 normal oral epithelia, 24 dysplastic oral leukoplakia (OLK), and 40 OSCC specimens, expressions of CXCL12 and CXCR4 were evaluated by immunohistochemistry., Results: CXCR4 was expressed in 37.5% of OLK and 60% of OSCC. CXCL12 was detected in 50% of OLK and 62.5% of OSCC. In OLK, CXCR4 positive ratio showed no significant difference from normal epithelia, but the CXCL12 positive ratio was significantly higher. Significant relationship between CXCL12 and CXCR4 was found both in OLK and OSCC., Conclusion: Our results indicated that CXCL12/CXCR4 axis may play roles from early steps of oral malignant transformation and contribute to the progress of oral carcinogenesis.

Oral carcinogenesis is a multistep process and requires accumulation and interplay of a series of molecular events. Chemokines and their receptors have been suggested to play important roles in the initiation or progression of cancers. Until now, no report focuses on their alterations in premalignant stage of oral squamous cell carcinoma (OSCC). Compared with normal tissues, mRNA levels of 9 chemokines and 3 chemokine receptors including CXCR7 in oral leukoplakia (OLK) were increased more than two folds by microarray analysis. Then, CXCR7 was selected for further confirmation and immunohistochemistry examination during multistage oral carcinogenesis. CXCR7 was expressed in 85% of OLK and 86% of OSCC. However, only 8% (1 of 13 cases) of normal tissue displayed CXCR7 immunostaining. The positive ratios of CXCR7, CXCL12 and CXCL11 in OLK and OSCC tissues respectively, were significantly higher than that in normal epithelia (P < 0.05), although no significant difference was found between OLK and OSCC. Meanwhile, CXCR7 always concomitantly expressed with it ligands in OLK and OSCC tissues. Our results indicated that CXCR7-CXCL12/CXCL11 axis might play important roles in oral carcinogenesis.

Amplification of chromosomal region 11q13 is one of the genetic alterations most frequently observed in oral squamous cell carcinoma (OSCC). Both TAOS1, a recently identified gene, and EMS1 were thought as two important target oncogenes for driving 11q13 amplification, and their contributions to oral carcinogenesis were hypothesized. Therefore we investigated amplifications of TAOS1 and EMS1 genes and their relations to clinicopathological variables in premalignant lesions (leukoplakias) and primary OSCC. TAOS1 amplification, beginning from mild-dysplastic epithelia, occurred in 33.3% of leukoplakias and 51.5% of OSCC. EMS1 amplification, beginning from moderate-dysplastic epithelia, occurred in 20% of leukoplakias and 57.6% of OSCC. Both gene amplifications were significantly related to different stages of oral carcinogenesis (p<0.05). During multistage carcinogenesis, no gene amplification was observed in normal tissue and non-dysplastic leukoplakias while, in OSCC with metastasis, amplification frequency increased significantly (p<0.005). Both TAOS1 and EMS1 amplifications were significantly associated with larger tumor size, presence of lymph node metastasis, poor histological differentiation and advanced clinical stage. Our data suggested potential roles in oral carcinogenesis and that TAOS1 might be involved earlier than EMS1. Both genes might be candidate biomarkers for diagnosis and prognosis in OSCC.